A wealth of data argues that mammalian prions are composed of infectious proteins designated PrPSc. Encouraged by investigations demonstrating the production of synthetic prions was possible by mutating the prion protein (PrP) gene, we chose to study a subset of recombinant (rec) PrPs folded into beta-rich structures. PrP amyloid is formed from N-terminally truncated PrPSc referred to as PrP 27-30, which comprises residues 89 to 230 in mouse (Mo) PrP and retains prion infectivity. Using only recMoPrP(89-230) produced in E. coli to form amyloid fibrils, we inoculated the fibrils intracerebrally into transgenic (Tg) mice expressing MoPrP(delta-23-88) (Supattapone et al. 2001). After more than 300 days, none of the mice had developed symptoms of CMS disease;therefore, we reported that the N-terminally truncated MoPrP(89-230) folded into amyloid fibrils were not infectious. Subsequently, we found that all of these mice developed neurologic dysfunction between 380 and 660 days after inoculation. Western blotting of brain extracts showed protease-resistant PrP and serial transmission of the prions to wild-type (wt) FVB and Tg(MoPrP)4053 mice gave incubation times of 150 and 90 days, respectively. Neuropathological examination suggests that a novel prion strain was created, one that is distinct from the widely used RML strain. Our discovery supports the propositions that prions are infectious proteins and that sporadic prion disease requires only the spontaneous conversion of PrPC into PrPSc and opens many new avenues of research. Based on these findings, we plan to produce wt and mutant sequences of mouse, hamster, human and bovine prions from recombinant PrPs produced in E. coli. Next, we shall determine conditions under which different prion strains can be created by modifying the PrP amyloid polymerization process. We shall utilize some of the protocols that have been successful in creating different strains of yeast prions. The different strains will be characterized by incubation times, distribution of neuropathological changes, PrP deposition profiles in brain, measurements by the conformation-dependent immunoassay, conformational stability assays, as well as glycoform and protease-resistant fragment size analysis. We plan to attempt to identify PrPSc-like molecules within recPrP amyloid. The likelihood of success for this investigation will depend on the abundance of PrPSc in the amyloid fibrils. We plan to characterize the structural features of recPrP amyloids producing different prion strains. We also propose to develop an amyloid-screening assay for recombinant PrPs produced in E. coli. If this can be done, then we shall be able to identify an array of mutant prions, the infectivity of which will be assessed by bioassay in Tg mice.